Project Summary: Rampant antibiotic resistance is predicted to cause devastating effects on world health over the next thirty years. The treatment of simple infections is becoming increasingly complicated by bacteria resistant to known antibiotics. The effects of resistance on healthcare are exacerbated by a lack of novel antibiotic development. In particular, Gram-negative bacteria are of increasing concern. All Gram-negative bacteria have an outer membrane (OM), the presence of which complicates the discovery of new antibiotics, acting as a barrier to therapeutics and rendering many current antibiotics useless. The OM, which is built by several molecular machines, is essential in Gram-negative bacteria. Each OM biogenesis machine requires at least one lipoprotein to function properly. Many lipoproteins are virulence factors, and several are essential to cellular processes. Gram-negative bacteria must, therefore, navigate the challenge of transporting lipoproteins from the inner membrane (IM) across the aqueous periplasm to the OM. The Lol system transports lipoproteins to the OM. LolCDE removes lipoproteins from the IM. LolA, a periplasmic chaperone, receives lipoproteins from LolCDE and transports them across the periplasm where they are inserted into the OM by LolB. Surprisingly, my lab recently found that lipoproteins can still reach the OM in the absence of LolAB. Functional lipoprotein transport in the absence of LolAB demonstrates that an alternate route of lipoprotein transport must exist. This finding challenges the current paradigm of lipoprotein transport, as the Lol system was previously thought to be the only mechanism by which lipoproteins reach the OM. Therefore, I hypothesize that an alternate lipoprotein transport pathway delivers lipoproteins to the OM. Using my lab?s ?lolAB strain of Escherichia coli, I am uniquely positioned to identify and define alternate routes of lipoprotein transport. I will use biochemical and genetic assays to test my hypothesis in two aims. In Aim 1, I will characterize the interaction of LolCDE with the alternate route of lipoprotein transport. Although lipoproteins are still transported to the OM in the absence of the LolAB pathway, my lab has confirmed that LolCDE is absolutely essential to all pathways of lipoprotein transport. I will mutate residues in LolCDE and test the function of the mutants in the alternate lipoprotein transport pathway. I will then test drug sensitivity to assess OM permeability in LolC mutants. OM biogenesis machine purification and membrane fractionation will be used to test the ability of mutants to transport lipoproteins. In Aim 2, I will identify and characterize genes important to the alternate lipoprotein transport pathway. I will use an unbiased global screen of transposon mutants to identify these genes. I will then use OM biogenesis machine purification and membrane fractionation to classify genes important to the alternate pathway. Together, these two aims will close a gap in our understanding of lipoprotein transport in Gram-negative bacteria. Due to the essentiality of lipoproteins and their transport to the OM, this research will provide important insight that will support future discovery of therapeutics targeting OM biogenesis.